Non-ribosome peptide-polyketone-polyamine bactericidal active substance Fcl-8 and application thereof

Abstract

The invention relates to a brand-new microbial natural metabolite, namely Fcl-8, which is derived from a fermentation solution of Xenorhabdius budapestensis XBD8, and is characterized in that the Fcl-8 is a new microbial natural metabolite, namely Fcl-8, which is derived from the fermentation solution of the Xenorhabdius budapestensis XBD8. The purified Fcl-8 is a snow white flocculent crystal and is very easy to dissolve in water, and the solid state of the purified Fcl-8 is as shown in Figure 1. The chemical structural formula of the Fcl-8 is as shown in a figure 2, the chemical formula is C57H102N14O12, the molecular weight is 1175.78744, and the typical secondary mass spectrum information of the Fcl-8 is as shown in a figure 3. The Fcl-8 is derived from a microbial natural metabolite, is novel in structure and high in biological activity, and has very high potential to be developed into a novel biopesticide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and in particular to a microbial natural metabolite and application thereof. BACKGROUND

[0002] Microbial natural metabolites, also known as secondary metabolites, in recent years, with the rapid development of genomics, metabolomics and bioinformatics, more and more natural products have been identified, some of which have been proven to be effective antibiotics and widely used, such as vancomycin, erythromycin, etc. Insect pathogenic nematode symbiotic bacteria are a model organism for studying symbiotic relationship, and have become a development hotspot in the field of biopharmaceutical research. Insect pathogenic nematode symbiotic bacteria are a kind of gram-negative bacteria of enterobacteriaceae, which parasitizes in the intestinal tract of insect pathogenic nematodes, including Xenorhabdus and Photorhabdus, which are symbiotic with Steinernema and Heterorhabditis respectively. At present, a variety of bioactive ingredients have been isolated and identified from known Xenorhabdus, which have novel structures, high biological activity and potential for developing new biological pesticides, such as insecticidal, antifungal, antibacterial, nematicidal, etc., and have great application prospect in the field of agriculture. According to literature reports, Xenorhabdus mainly has the following types of bioactive compounds:

[0003] (1) Disulfide pyrroles: this kind of substance is isolated from Xenorhabdus nematophilus, which has good effect on a variety of pathogenic bacteria, especially gram-positive bacteria, mainly inhibiting the synthesis of bacterial RNA and thus affecting the synthesis of protein.

[0004] (2) Indole derivatives: this kind of compound has good control effect on gram-positive and gram-negative bacteria, and has obvious inhibitory effect on bacterial growth. The mechanism of action is mainly to increase the synthesis of nucleotide ppGpp, and inhibit the synthesis of RNA of gram-negative and gram-positive bacteria.

[0005] (3) Bacteriocins: bacteriocins have a narrow antibacterial spectrum, and usually only inhibit the growth of strains with close genetic relationship. Under general conditions, the expression amount in bacteria is very low.

[0006] (4) Amide compounds: two amide compounds, cyclobutanediolactam and oxalylamide, are isolated from Xenorhabdus nematophilus YL001 strain, which have good inhibitory effect on the mycelial growth of Botrytis cinerea.

[0007] (5) Water-soluble benzopyrene (XCNs): water-soluble isocoumarins are the main bacteriostatic compounds in Xenorhabdus, which have strong inhibitory effect on Pseudomonas cucumis, Pseudomonas ramorum, Pseudomonas capsici and other bacteria. They have a wide range of gram-positive and gram-negative bacteria.

[0008] (6) Peptides, most of which have strong alkaline, thermal stability and broad-spectrum antibacterial spectrum. In Xenorhabdus bacteria F1 strain, a family of lysine-rich cyclic lipopeptides (PAX) consisting of 5 components is isolated, which not only has strong inhibitory activity on plant and human pathogenic fungi, but also has certain inhibitory effect on bacteria and yeast, and has no toxicity to hamsters and insect cells.

[0009] Among them, a class of fabclavine, the English name is fabclavine, is a natural product identified in the fermentation broth of insect pathogenic symbiotic bacteria Xenorhabdus spp. Research has found that fabclavine has broad-spectrum resistance to gram-positive and gram-negative bacteria, pathogenic fungi, mycoplasma, nematodes and other organisms , and has great development potential in medical and agricultural fields. At present, most of the researches on fabclavine are related to its biological activity and synthesis mechanism, and there are few related researches and inventions of industrialization development. SUMMARY

[0010] The present application first provides a brand new microbial natural metabolite: Fcl-8, which is derived from Xenorhabdus budapestensis XBD8 fermentation broth and is one of the main active ingredients of XBD8. The purified Fcl-8 is snow-white flocculent crystal, which is easily soluble in water, and its solid state is as shown in Figure 1 . The chemical structural formula of Fcl-8 is as shown in Figure 2 :

[0011] ,

[0012] The chemical formula of Fcl-8 is C 57 H 102 N 14 O 12 , the molecular weight is 1175.78744, and the typical secondary mass spectrum information is as shown in Figure 3 .

[0013] The Xenorhabdus budapestensis XBD8 strain, i.e., the XBD101 strain, is a Steinernema diaplei symbiotic bacterium isolated from soil samples collected in the northeast by the inventors' laboratory. The XBD101 strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No: 22056. The functions and applications of the strain are recorded in Chinese invention patent ZL202110766306.8, which has been granted a patent (CN113265364B), and the disclosure thereof is incorporated herein by reference. In addition, the XBD8 strain mentioned in the text of the present application is the XBD101 strain.

[0014] The natural product Fcl-8 obtained by the inventors from the fermentation broth of XBD8 after purification and purification is identified as a non-ribosomal peptide-polyketide-polyamine compound. Experiments have verified that it has a significant inhibitory effect on a variety of plant pathogenic bacteria and plant pathogenic fungi, including but not limited to Xanthomonas campestris, Erwinia amylovora, Rhizoctonia solani, Fusarium oxysporum, Alternaria solani, Sclerotinia sclerotiorum, Fusarium graminearum, Colletotrichum orbiculare, Fusarium pseudograminearum, Rhizoctonia cerealis, Verticillium dahliae, etc. Fcl-8 is derived from microbial natural metabolites, has a novel structure, high biological activity, and has the potential to be developed into a new type of biological pesticide. As can be seen from the experimental verification, Fcl-8 with a lower concentration (such as 3.91 ppm) has a good inhibitory effect on plant pathogenic bacteria, and its inhibitory effect is significantly improved as the concentration increases; for plant pathogenic fungi, 5 mg / L (i.e., 5 ppm) of Fcl-8 concentration also has a significant inhibitory effect, so in actual application, Fcl-8 with a concentration of more than 5 mg / L is preferably prepared into a biological pesticide, such as a biological fungicide, which can achieve stable and effective technical effects without environmental pollution. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A solid state diagram of purified Fcl-8;

[0016] Figure 2 Fcl-8 chemical structure;

[0017] Figure 3 Fcl-8 secondary mass spectrum information chart;

[0018] Figure 4-1 Fcl-8 MIC against Xanthomonas campestris;

[0019] Figure 4-2 Fcl-8 MIC against Erwinia amylovora;

[0020] Figure 4-3 Fcl-8 MIC against Xanthomonas campestris;

[0021] Figure 5 Fcl-8 inhibitory effect on the mycelial growth of 9 plant pathogenic fungi. DETAILED DESCRIPTION

[0022] The application will be further described below in conjunction with the examples, but in no way limits the application, and for those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the application shall be included in the protection of the application.

[0023] Example 1:

[0024] Fcl-8 is derived from Xenorhabdus budapestensis XBD8 fermentation broth, and is one of the main active ingredients of XBD8. The XBD8 strain is XBD101, which is a Steinernema diaplei symbiotic bacteria isolated from soil samples collected in the northeast by the laboratory. The XBD8 strain is independently isolated by the laboratory, and has high antagonistic effect on various plant pathogenic fungi and pathogenic bacteria.

[0025] The inventors analyzed the XBD8 fermentation broth after purification, and obtained a new natural product: Fcl-8, which is a non-ribosomal peptide-polyketide-polyamine bactericidal active substance. The specific separation and purification steps are as follows:

[0026] The XBD8 strain stored in the laboratory was taken out from the-80℃ refrigerator, streaked on LB (containing 100 μg / mL Amp) solid plate, and cultured in a 28℃ incubator for 48 h;

[0027] Pick a single colony and inoculate in 3 mL of LB (containing 100 μg / mL Amp) liquid medium, and cultivate at 28℃, 200 r / min for 18 h as a primary seed liquid;

[0028] The first-stage seed liquid was transferred to a shake flask containing 100 mL of LB liquid medium, and cultured at 28°C and 200 r / min for 18 h as a second-stage seed liquid.

[0029] The second-stage seed liquid was inoculated into a 1000 mL / 2000 mL shake flask at an inoculation amount of 1%, and fermented for 48 h.

[0030] The fermentation liquid was centrifuged at low speed (4500 rpm) and low temperature (4°C) to remove the bacterial cells, and the supernatant was collected and adsorbed on HP-20 resin overnight. The adsorbed resin was eluted twice with 30% acetonitrile. The eluate was rotary evaporated to obtain the eluted concentrated liquid. The concentrated liquid was pre-cooled at -80°C and dried overnight using a low-temperature vacuum drying instrument. The dried sample was quickly dissolved in 100% methanol, washed, centrifuged to obtain a precipitate, and the precipitate was dissolved in water to obtain a pretreated sample.

[0031] The pretreated sample was purified by HPLC. Instrument: Agilent 1260; chromatographic column: Durashell C18 (L) ; mobile phase: A-acetonitrile; B-0.1% trifluoroacetic acid solution (0 min-22% A, 10 min-22% A, 11 min-90% A, 17 min-90% A, 18 min-22% A, 26 min-22% A), flow rate: 1.0 ml / min; column temperature: 35°C; detection wavelength: UV-203 nm.

[0032] The purified Fcl-8 was a snow-white flocculent crystal, which was easily soluble in water. The solid state thereof is shown in Figure 1 . The chemical structural formula of Fcl-8 is shown in Figure 2 . The chemical formula is C 57 H 102 N 14 O 12 , the molecular weight is 1175.78744, and the typical secondary mass spectrum information is shown in Figure 3 . The mass spectrometry detection conditions are as follows:

[0033] Instrument: LC-MS (ion / high performance liquid chromatography-high resolution mass spectrometer, Q Exactive); column: Hypersil GOLD™ VANQUISH (100 x 2.1, 1.9um, Thermo), Method duration: 14.00 min; Runtime: 0 to 14 min; Polarity: positive; Default charge: 1; Inclusion: on; Resolution: 17500; AGC target: 2e5; Maximum IT: 100ms; Isolation window: 2.0m / z; (N)CE / stepped nce: 20, 40.

[0034] Example 2: MIC of Fcl-8 on bacteria

[0035] The MIC (minimum inhibitory concentration) method in GB YY / T0688.1-2008 was used to detect the MIC of Fcl-8 on three typical plant pathogenic bacteria, Xanthomonas campestris, Erwinia amylovora and Rhizoctonia solani. The specific steps are as follows:

[0036] 1) Use a loop to dip Xanthomonas campestris, Erwinia amylovora and Rhizoctonia solani bacterial liquid on ordinary LB plate, and incubate in 28℃ incubator for 12-16 h.

[0037] 2) Pick single colonies of the three strains in LB liquid medium, and inoculate again at 1% inoculation amount after overnight culture at 200 rpm / min.

[0038] 3) Take the three strains to measure their OD values 2-3 hours after transfer 600 , dilute the bacterial liquid OD 600 to 0.5 when the OD 600 value reaches 0.8-1.0.

[0039] 4) Take 20 μL of bacterial liquid with OD 600 0.5, add to a 96-well plate containing 180 μL of Fcl-8 with different concentrations (concentrations: 250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm, 15.63 ppm, 7.83 ppm, 3.91 ppm, 1.95 ppm, 0.98 ppm, 0.49 ppm) obtained by double dilution, three replicates for each concentration, mix well, and incubate in 28℃ incubator for 24 h. Then use the enzyme marker to measure the OD 600 of the three strains under different treatments, and the minimum concentration of antibacterial peptide that can obviously inhibit the growth of the bacterial liquid is MIC.

[0040] The inhibitory effect of Fcl-8 on Xanthomonas campestris, Erwinia amylovora, and Alternaria dauci is shown in Figure 4 (including Figure 4-1 to Figure 4-3 ) and the MICs thereof are 7.81 mg / L, 1.95 mg / L, and 3.91 mg / L, respectively, and the inhibitory effect is very significant.

[0041] Example 3: Inhibitory effect of Fcl-8 on fungi

[0042] The inhibitory effect of Fcl-8 on the mycelial growth of nine plant pathogenic fungi, Rhizoctonia solani, Fusarium oxysporum, Alternaria solani, Sclerotinia sclerotiorum, Fusarium graminearum, Colletotrichum orbiculare, Fusarium pseudograminearum, Rhizoctonia cerealis, and Verticillium dahliae, was determined by the growth rate method.

[0043] 1) The pathogenic fungi stored at -80°C were inoculated on PDA plates and incubated at 25°C in the dark for 5 days.

[0044] 2) Fcl-8 was added to PDA medium to prepare toxin-containing plates with concentrations of 5 ppm, 10 ppm, and 20 ppm, and the plates were mixed and poured into 6 cm plates. An equal amount of water was used as a blank control, and each treatment was repeated three times.

[0045] 3) The activated pathogenic fungi were inoculated on toxin-containing plates with different concentrations of Fcl-8 and incubated at 25°C in the dark for 5 days.

[0046] 4) The colony diameters under each treatment were measured using the cross method, and the mycelial growth inhibition rates of each target fungus were calculated according to Formulas (1) and (2) as percentages (%), with the results rounded to two decimal places.

[0047] Formula (1): D = D1 - D2

[0048] Formula (2):

[0049]

[0050] In Formula (1), D: colony growth diameter; D1: colony diameter; and D2: mycelial cake diameter.

[0051] In formula (2): I: mycelium growth inhibition rate; D0: blank control colony growth diameter; Dt: colony growth diameter of different treatments.

[0052] The inhibition effect of Fcl-8 on the mycelium growth of nine plant pathogenic fungi such as Rhizoctonia solani, Fusarium oxysporum, Alternaria solani, Sclerotinia sclerotiorum, Fusarium graminearum, Colletotrichum orbiculare, Fusarium pseudograminearum, Rhizoctonia cerealis and Verticillium dahliae is shown in Table 1 and Table 1. Figure 5 The inhibition rate of 5mg / L Fcl-8 on different fungi is between 20.7±1.7% and 92.3±1.8%, the inhibition rate of 10mg / L Fcl-8 on different fungi is between 58.6±4.8% and 96.9±1.8%, and the inhibition rate of 20mg / L Fcl-8 on different fungi is between 71.6±1.2% and 100.4±1.6%, which is very significant.

[0053]

[0054] From the above experiments, it can be seen that Fcl-8 with a lower concentration (such as 3.91ppm) has a good inhibitory effect on plant pathogenic bacteria, and the inhibitory effect is significantly improved with the increase of concentration; for plant pathogenic fungi, 5mg / L (5ppm) Fcl-8 concentration also has obvious inhibitory effect, so in practical application, Fcl-8 with a concentration of more than 5mg / L is preferably prepared into a biological pesticide, such as a biological fungicide, which can achieve stable and effective technical effects without environmental pollution.

[0055] The above-described embodiments are only preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art should fall within the protection scope of the claims of the present application.

Claims

1. A non-ribosomal peptide-polyketide-polyamine class bactericidal active substance Fcl-8, characterized by, The chemical structure of Fcl-8 is shown as follows: ， Chemical Formula: C 57 H 102 N 14 O 12 Molecular Weight: 1175.78744.

2. The Fcl-8 according to claim 1, characterized in that, The typical mass spectrum information of Fcl-8 is shown in Figure 3.

3. The Fcl-8 of claim 1, wherein, The Fcl-8 is snow white flocculent crystal, very easy to dissolve in water.

4. The Fcl-8 of claim 1, wherein, The Fcl-8 is purified from the fermentation broth of Xenorhabdus budapestensis XBD8; the XBD8 strain is XBD101 strain, which is preserved in China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No:22056.

5. The Fci-8 according to any one of claims 1-4, characterized in that, The Fcl-8 has significant inhibitory effect on plant pathogenic bacteria.

6. The Fcl-8 of claim 5, wherein, The plant pathogenic bacteria include but are not limited to Xanthomonas campestris, Erwinia amylovora, Rhizoctonia solani.

7. The Fci-8 according to any one of claims 1-4, characterized in that, The Fcl-8 has significant inhibitory effect on plant pathogenic fungi.

8. The Fci-8 of claim 7, wherein, The plant pathogenic fungi include but are not limited to Rhizoctonia solani, Fusarium oxysporum, Alternaria solani, Sclerotinia sclerotiorum, Fusarium graminearum, Colletotrichum orbiculare, Fusarium pseudograminearum, Rhizoctonia cerealis and Verticillium dahliae.

9. Use of Fcl-8 in any one of claims 1-8 in the preparation of a biopesticide.

10. Use according to claim 9, characterized in that, The concentration of Fcl-8 is above 5 mg / L.

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